Development of a highly sensitive colloidal gold semiquantitative method for the determination of difenoconazole residues in citrus.

Introduction: Difenoconazole (DIFE) is a common pesticide used in citrus cultivation; excessive intake can cause neurological damage to the organism, and the existing colloidal gold immunochromatographic test strips cannot meet the requirements for the detection of citrus samples. Methods: Difenoconazole test strip was prepared based on the colloidal gold immunochromatographic technique (GICT), and its application in citrus samples was investigated; with colloidal gold (CG) as the probe, the optimization of GICT parameters, and the determination of reaction method, the immunochromatographic test strips for the detection of DIFE in citrus was developed, and the limit of detection (LOD), specificity, accuracy, and stability of the test strips were verified. Results: The results showed that the visual detection limit of the prepared colloidal gold immunochromatographic test strips was 0.2 mg/kg and the quantitative range was 0.06-0.6 mg/kg, and the test strips could specifically identify DIFE and have no cross-reaction with other common triazole pesticides. The detection method established in this study was verified by the GC-MS method, and the detection results achieved good consistency (R2 > 0.98). Conclusion: The test strips developed in this study have good performance and can be used for highly sensitive detection of citrus samples.

Establishment of a Colloidal Gold Immunochromatographic Method for the Detection of Cypermethrin in Tobacco.

In this study, the establishment of a colloidal gold immunochromatographic method for the detection of cypermethrin in tobacco was achieved by using colloidal gold immunochromatography: strong specificity and high sensitivity of cypermethrin semi-antigens and encapsulants were prepared during the study. The best colloidal gold solution was prepared by spectrophotometer and transmission electron microscope screening; the preparation process of gold-labeled antibodies was optimized, and finally the product of colloidal gold rapid detection test strips for cypermethrin was developed. The results of technical parameters and detection indexes showed that the detection limit of cypermethrin in tobacco was 1 mg/kg, and there was no cross-reaction with bifenthrin, cypermethrin, cyfluthrin and phenothrin, and the detection results of 30 tobacco samples were consistent with those of gas chromatography.

Hapten designs based on aldicarb for the development of a colloidal gold immunochromatographic quantitative test strip.

The common carbamate insecticide aldicarb is considered one of the most acutely toxic pesticides. Herein, rational design was used to synthesize two haptens with spacers of different carbon chain lengths. The haptens were then used to immunize mice. The antibodies obtained were evaluated systematically, and a colloidal gold immunochromatographic strip was developed based on an anti-aldicarb monoclonal antibody. The 50% inhibition concentration and linear range of anti-aldicarb monoclonal antibody immunized with Hapten 1 were 0.432 ng/mL and 0.106-1.757 ng/mL, respectively. The cross-reactivities for analogs of aldicarb were all <1%. The limit of detection of the colloidal gold immunochromatographic strip was 30 µg/kg, and the average recoveries of aldicarb ranged from 80.4 to 110.5% in spiked samples. In the analysis of spiked samples, the test strip could accurately identify positive samples detected by the instrumental method in the GB 23200.112-2018 standard but produced some false positives for negative samples. This assay provides a rapid and accurate preliminary screening method for the determination of aldicarb in agricultural products and environments.

Rapid screening of 70 colorants in dyeable foods by using ultra-high-performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry with customized accurate-mass database and mass spectral library.

A rapid screening method of 70 colorants for regulatory control in dyeable foods was established using ultra-high-performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q/Orbitrap MS) with customized accurate-mass database and mass spectral library. A rapid, high-throughput, and simple sample pretreatment condition with low reagent consumption and high recovery was developed on the basis of ultrasound-assisted extraction and dispersion solid-phase extraction. Rapid screening was conducted by comparing the experimentally measured exact mass of the parent and fragment ions, the isotope pattern, and the retention time with the accurate-mass database and by matching the acquired MS/MS spectra against the mass spectral library. The performance of the method was evaluated in terms of linearity, limits of detection, limits of quantitation, recovery, repeatability, reproducibility, and matrix effect. The proposed method was applied for simultaneous analysis of 70 colorants in seven kinds of dyeable foods, and it exhibited great potential for broad, sensitive, and reliable.

Effects of electrolyzed water treatment on pesticide removal and texture quality in fresh-cut cabbage, broccoli, and color pepper.

The effects of alkaline and acidic electrolyzed water (AlEW, AcEW) treatment on the removal of pesticides (phorate, chlorpyrifos, lambda-cyhalothrin, cyfluthrin, procymidone, and chlorothalonil) and texture quality of fresh-cut cabbage, broccoli, and color pepper were investigated. AlEW efficiently removed pesticides from color pepper, whereas AcEW was the optimal treatment for pesticide removal from cabbage and broccoli. AcEW resulted in greater losses of pyrethroid and organophosphates than fungicides, while AlEW was superior for removing fungicides. The best pesticide removal from cabbage (72.28%-91.04%) was achieved by continuous oscillation treatment, while intermittent oscillation for 20 min achieved optimal results for broccoli and color pepper (72.28%-90.11% and 72.24%-88.12%, respectively). No significant deterioration in texture was detected in samples treated with electrolyzed water for 5-25 min. The results suggest that electrolyzed water treatment is effective for removing organophosphate, pyrethroid, and fungicide residues from fresh-cut vegetables while not negatively affecting their texture quality.

Extensive screening of cyclopeptide toxins in mushrooms by ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry.

A non-target screening method of cyclopeptide toxins and their analogues in mushroom was developed, using ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) followed by mass spectrometry databases retrieval and software tools analysis for the candidate analogues. Three cyclopeptide toxins in the toxic mushroom Amanita rimosa were firstly screened without standard, and two of them were unknown analogues which were tentatively identified by the accurate masses, isotopic patterns and characteristic fragments. A validated quantitative method was performed to rapidly quantify three major cyclopeptide toxins in the Amanita rimosa sample including α-manitin, ß-amanitin and phalloidin, and their contents were detected to be 4.52 mg/kg, 2.37 mg/kg and 2.53 mg/kg, respectively. The developed method has good selectivity and sensitivity for rapid and comprehensive screening the cyclopeptide toxins and their analogues in mushrooms at trace levels. Successful non-target screening of trace cyclopeptide toxin analogues will guarantee the food safety in mushrooms consumption.

The Epidemiology of Monophasic Salmonella Typhimurium.

Salmonella enterica remains an important foodborne pathogen in all regions of the world, with Typhimurium as one of the most frequent serotypes causing foodborne disease. However, the past two decades have seen a rapid worldwide emergence of a new Salmonella serotype, namely monophasic variant of S. Typhimurium, whose antigenic formula is 1,4,[5],12:i:-. It has become one of the 2-5 most common Salmonella serotypes responsible for animal and human infections in different regions. The global epidemic of monophasic S. 1,4,[5],12:i:- has mainly been characterized by an increase in multidrug-resistant S. 1,4,[5],12:i:- isolated in Europe since 1997. The unexpected link to swine has escalated monophasic S. Typhimurium infections to the status of a global public health emergency. The large-scale application of whole genome sequencing (WGS) in the last 10 years has revealed the phylogenetic associations of the bacterium and its antimicrobial resistance (AMR) genes. Local and global transmission reconstructed by WGS have shown that different clones have emerged following multiple independent events worldwide, and have elucidated the role of this zoonotic pathogen in the spread of AMR. This article discusses our current knowledge of the global ecology, epidemiology, transmission, bacterial adaptation, and evolution of this emerging Salmonella serotype.

Rapid detection of trace Salmonella in milk and chicken by immunomagnetic separation in combination with a chemiluminescence microparticle immunoassay.

Rapid detection of trace Salmonella is urgently needed to ensure food safety. We present an innovative pretreatment strategy, based on a two-step enrichment culture and immunomagnetic separation, combined with a chemiluminescence microparticle immunoassay to detect at least one proliferative Salmonella cell in 25 mL (25 g) food. The capture performance of immunomagnetic beads (IMBs) of sizes for Salmonella was investigated, and the IMBs of size 2.8 µm showed a high capture efficiency of 60.7% in 25 mL milk and 74.5% in 25 mL chicken culture filtrate, which ensured the successful capture of trace Salmonella after 2.5 h in situ enrichment even from only one Salmonella cell. The separated Salmonella cells, reaching an amount of 103 colony-forming units (CFU) by a secondary enrichment for 3 h, were detected by a horseradish peroxidase chemiluminescence reaction with 4-(1-imidazolyl)phenol as an enhancer, which evidenced a linear response for Salmonella concentrations ranging from 2.3 × 102 to 7.8 × 104 CFU/mL. The entire detection process was completed within 8 h, with a very low detection limit of 1 CFU/25 mL (25 g), which was verified by colony counting, and a small degree of interference of 0.17-1.06%. Trace Salmonella from five different serovars in milk and chicken was successfully detected without false negative or false positive results. Furthermore, this study provides a basis to develop a fully automated instrument based on IMBs that includes all steps from sample preparation to chemiluminescence microparticle immunoassay for high-throughput screening of foodborne pathogens. Graphical abstract.

Detection of plasmid-mediated tigecycline-resistant gene tet(X4) in Escherichia coli from pork, Sichuan and Shandong Provinces, China, February 2019.

The plasmid-mediated high-level tigecycline resistance gene, tet(X4), was detected in seven Escherichia coli isolates from pork in two Chinese provinces. Two isolates belonged to the epidemic spreading sequence type ST101. Tet(X4) was adjacent to ISVsa3 and concurrent with floR in all seven isolates. In addition to IncFIB, the replicon IncFII was found to be linked to tet(X4). This report follows a recent detection of tet(X3)/(X4) in E. coli from animals and humans in China.

Microbial composition of spoiled industrial-scale Sichuan paocai and characteristics of the microorganisms responsible for paocai spoilage.

The microorganisms of spoiled industrial-scale Sichuan paocai (ISSP) were isolated using six types of media, and 16S rRNA and 26S rRNA gene sequence analyses were used to identify the isolates. Meanwhile, the microbial composition was investigated using a culture-independent method through 16S rRNA and ITS sequencing on an Illumina MiSeq platform. The results obtained by these two methods were compared. Furthermore, characteristics of the isolated microorganisms responsible for ISSP spoilage were studied. Sixty-two strains belonging to twenty-three species, including three ammonia-producing genera, two gas-producing genera, two pectinase-producing genera, two cellulase-producing genera, three film-producing genera and one slime-producing genus, were isolated. Lactobacillus, Bacillus, Debaryomyces and Kazachstania were the dominant genera as confirmed through both culture-dependent and culture-independent methods. Bacillus, Paenibacillus, Pichia and Debaryomyces could be the main microorganisms responsible for ISSP spoilage. Bac. licheniformis was closely correlated with the off-flavour of ISSP. Pae. peoriae, Bac. stratosphericus, Bac. toyonensis and Bac. cereus were responsible for tissue softening, and Bac. subtilis, Bac. methylotrophicus, Pic. membranifaciens and Deb. hansenii caused film formation.

Rapid measurement of free cyanide in liquor by ion chromatography with pulsed amperometric detection.

This study investigated the measurement of free cyanide in liquor by ion chromatography coupled with pulsed amperometric detection (IC-PAD). Eluent concentration, interferent evaluation and method performance were discussed. Results show that free cyanide in liquor can be rapidly determined by the optimised IC-PAD method. A sample requires only 1:100 dilution and simple filtration before being subjected to IC-PAD. The linear range is 1-5000 µg/L with an R value of 0.9998. The detection limit is 1 µg/L for a 25 µL injection loop. The overall relative standard deviation (RSD) of the method is less than 5%, and the recovery range is from 98.1% to 105.0%. This study has been proven significant and may have potential applications in liquors analysis.

A sensitive and selective chemosensor for ascorbic acid based on a fluorescent nitroxide switch.

Ascorbic acid (AsA), also known as vitamin C, is a vital small-molecule antioxidant with multiple functions in vivo. It's the major natural antioxidant found in plants and is also an essential component of human nutrition. AsA plays a key role in many diseases-related biological metabolism. Therefore, sensitive and selective detection of AsA is greatly important in pharmaceutical, clinical and food industry. Here a sensitive and selective sensor for ascorbic acid detection based on the recovered fluorescence of NAPS-NO (N-propyl-triethoxysilane-4-(4-ylamino-1-oxy-2,2,6,6-tetramethylpiperdine)- naphthalimide) probe is described. The fluorescence of the naphthalimide moiety of NAPS-NO is inhibited by the nitroxide group, which is covalently linked to the fluorophore. Then, ascorbic acid reacts rapidly with the nitroxide moiety of NAPS-NO to form hydroxylamine, and the fluorescence properties of the naphthalimide moiety are recovered and the ESR signal decayed. Over a wide range from 80 nM to 50 µM, a good linear relationship between the fluorescence intensity and the concentration of ascorbic acid was found and the detection limit was estimated to be as low as 20 nM. To confirm the practical usefulness of the fluorophore-nitroxide probe, we demonstrated the use of NAPS-NO for the measurement of AsA in human blood serum and also successfully determined the concentration of AsA in HEK 293 cell lysate. Results from confocal laser scanning microscopy experiments demonstrated that this chemosensor is cell permeable and can be used as a fluorescent probe for monitoring ascorbic acid in living cells.

[Determination of streptomycin and dihydrostreptomycin residues in tomato paste by tandem dual solid phase extraction column-liquid chromatography-tandem mass spectrometry].

The method was specifically developed for the simultaneous determination of streptomycin and dihydrostreptomycin residues in tomato paste by tandem dual solid phase extraction (SPE) column cleanup-liquid chromatography-tandem mass spectrometry. The residues were extracted from the samples with phosphate buffer solution (pH 4). The cleanup was performed by the way of dispersive solid phase extraction and tandem dual solid phase extraction column. The polar chromatographic column was used to complete the separation of the analytes under gradient elution and the analytes were detected in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI +). The external standard calibration curves were used for the quantification. The linear ranges were from 0.01 to 0.2 mg/L with a good linear relationship (r > 0.999) for streptomycin and dihydrostreptomycin. The limit of quantification (LOQs) was 0.02 mg/kg for the both analytes. The recovery range was from 71% to 101% with the relative standard deviations (RSDs) between 2.3% and 15%. It was indicated that this method is accurate, easier, more sensitive, and has a better purification effect in the monitoring and analysis. The method is accurate and specific to monitor and analyze of streptomycin and dihydrostreptomycin residues in tomato paste and its products.

Indirect competitive enzyme-linked immuno-sorbent assay (ELISA) for nitroimidazoles in food products.

Ronidazole was used as the starting material to prepare an immunogen and coating antigen. An anti-nitroimidazole monoclonal antibody was produced and an indirect competitive ELISA was established to detect nitroimidazole compounds in food products. The IC(50) values were determined to be 0.20 ng/ml for metronidazole, 4.0 ng/ml for tinidazole, 0.17 ng/ml for dimetridazole and 0.24 ng/ml for ornidazole. Considering that nitroimidazoles were commonly used as veterinary drugs, nitroimidazole residues in food products of animal origin were detected by the method. The coefficient of variation for nitroimidazoles determination in contaminated chicken, chicken liver and shrimp were all <14% and the recovery rate was in the range 74.0-90.6%. The results proved that the developed method was successful in detecting nitroimidazoles in food products.

Generation of anti-trenbolone monoclonal antibody and establishment of an indirect competitive enzyme-linked immunosorbent assay for detection of trenbolone in animal tissues, feed and urine.

Trenbolone (TRE) is a steroid used by veterinarians on livestock to increase appetite and body weight. The use of TRE has been restricted because of its harmful side effect for consumers. To effectively control TRE residue in food and food product, a rapid and convenient immunoassay was developed by preparing an anti-TRE monoclonal antibody. The immunogen and coating antigen were prepared by coupling TRE hapten with carrier proteins via 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) method. The optimized method gave an average IC(50) value of 0.323 ng mL(-1) towards TRE and an average detection limit (LOD) of 0.06 ng mL(-1), which is much lower than the maximum residue levels (2.0 ng g(-1)) accepted by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The specificity of the antibody was evaluated by measuring cross-reactivity of six structurally related compounds, including 19-nortestosterone (9.7%), testosterone (0.13%), methyltestosterone (<0.01%), methandrostenolone (<0.01%), (+)-dehydroisoandrosterone (<0.001%) and ß-estradiol (<0.001%). The recovery rates of the test in detection of TRE-fortified animal tissue, urine and animal feed samples were in the range of 81.3-89.4%, while the intra- and inter-assay coefficients of variation were less than 12.0%.

Preparation of anti-Sudan red monoclonal antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of Sudan red in chilli jam and chilli oil.

Sudan dyes are banned to be used in food additives because of the carcinogenicity of their metabolites. A rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the residues of Sudan dyes. Novel immunogen and coating antigen were synthesized via glutaraldehyde linking. The hapten-bovine serum albumin (BSA) was applied as immunogen and the hapten-ovalbumin (OVA) was served as coating antigen. The monoclonal antibody obtained showed high sensitivity to Sudan I with an IC(50) value of 1.7 µg L(-1) in buffer and was suitable to detect the residues of Sudan red in food products. The specificity of the assay was studied by measuring cross-reactivity of the antibody with the structurally related compounds of Sudan II (<1%), Sudan IV (<1%) and para red (120%). Chilli jam and chilli oil samples spiked with Sudan dyes were analyzed by the method. The detection limit (LOD) of the ELISA method applied in chilli jam and chilli oil was 9.0 µg L(-1) and 19.6 µg L(-1), respectively. The recovery rates of Sudan-I in chilli oil and chilli jam were in the range of 80%-110% with coefficients of variation <25%. The intra-assay variation and inter-assay variation in buffer were both <9%.

Preparation of monoclonal antibody for melamine and development of an indirect competitive ELISA for melamine detection in raw milk, milk powder, and animal feeds.

Melamine (MEL) has been involved in several food recalls after the discovery of severe kidney damages in children and pets poisoned by melamine-adulterated food. To detect MEL residue in foods and animal feeds, an indirect competitive ELISA (cELISA) method was developed in this study based on preparation of monoclonal antibodies (MAbs) to MEL. The immunogen was prepared by linking MEL hapten with carrier protein via carbodiimide method. The method is applicable in the range of 5.0-135.0 microg L(-1) MEL in buffer solution, with an IC(50) value of 22.6 +/- 1.9 microg L(-1). The MAbs showed high specificity with low cross-reactivity (< or =1%) toward cyanurate, ammelide, and ammeline. The method was utilized in the detection of MEL in raw milk, milk powder, and animal feeds, with detection limits of 0.1 mg L(-1) for milk, 0.2 mg kg(-1) for milk powder, and 0.5 mg L(-1) for feeds. The recovery ratio was 79-110% for all matrices. The intra-assay and interassay coefficients of variation were <12.0 and <13.0%, respectively. Finally, the application of the cELISA in quantity evaluation of MEL in various feeds from local markets was evaluated and discussed.

Preparation of a monoclonal antibody and development of an indirect competitive ELISA for the detection of chlorpromazine residue in chicken and swine liver.

BACKGROUND: Chlorpromazine is a typical antipsychotic drug used to make food-producing animals calm and promote growth as feed additives. Accumulation of chlorpromazine in animal bodies would cause side effects in the circulatory and nervous systems, and have adverse effects on blood cells, the skin and the eye. To detect the chlorpromazine residue in food producing animals, an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed based on preparation of an anti-chlorpromazine monoclonal antibody. RESULTS: The antibody generated from immunogen of cationic bovine serum albumin (cBSA) coupled with chlorpromazine showed high sensitivity toward chlorpromazine with an IC(50) value of 0.73 ppb. The ELISA method was applied to detect swine liver and chicken samples spiked by chlorpromazine and satisfactory results were obtained. The recovery rates in chicken and swine liver were in the range of 88-95% and 86-95%, respectively; the intra-assay coefficients of variation were both < 15.3% and < 13.5%, respectively. CONCLUSION: An indirect competitive ELISA method based on a monoclonal antibody towards chlorpromazine with excellent sensitivity and specificity has been successfully developed. The immunoassay provided in this study was a hopeful alternative to chromatography spectrometry for regulatory analysis of chlorpromazine residue in food-producing animals.

Developing and optimizing an immunoaffinity cleanup technique for determination of quinolones from chicken muscle.

An immunoaffinity chromatographic method was developed using an antibody mediated immunosorbent to selectively extract and purify 10 quinolones (marbofloxacin, norfloxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, difloxacin, sarafloxacin, oxolinic acid, and flumequine) in chicken muscle followed by HPLC. The operating conditions of the immunoaffinity chromatography (IAC) column were optimized, and the IAC has been successfully used for the isolation and purification of 10 quinolones from chicken muscle tissue. The optimized immunoaffinity column sample cleanup procedure combined with HPLC coupling to fluorescence detection afforded low limits of detection (0.1 ng g(-1) for danfloxacin and 0.15 ng g(-1) for all other quinolones tested). The method was also applied to determine quinolone residues in commercial muscle samples.

Elevated levels of gremlin-1 in eutopic endometrium and peripheral serum in patients with endometriosis.

OBJECTIVE: To examine the expression of gremlin-1 (GREM1) on the levels of messenger RNA (mRNA) and protein in eutopic endometrium and its serum level in patients with endometriosis. DESIGN: Prospective, experimental study using reverse-transcription polymerase chain reaction, Western blot, immunofluorescence, and ELISA. SETTING: Gynecological oncology laboratory in a department of obstetrics and gynecology in a medical college in China. PATIENT(S): Thirty-five patients with endometriosis and 23 healthy control women. INTERVENTION(S): During surgery, the eutopic endometria and peripheral serum were obtained from the patients with endometriosis and the control women. MAIN OUTCOME MEASURE(S): The cellular compartment location of GREM1 expression was examined by using immunofluorescent double staining. The expression levels of mRNA and protein for GREM1 were determined by reverse-transcription polymerase chain reaction and Western blot, respectively. The serum level of GREM1 was measured by indirect ELISA. RESULT(S): The expression of GREM1 was defined within endometrial blood vessel endothelium exclusively, with the concomitant expressions of GREM1 and CD146. The expression of GREM1 on the levels of mRNA and protein was significantly higher in eutopic endometria of patients with endometriosis than in those from healthy control women. According to the ELISA established in our laboratory, the concentration of GREM1 in peripheral serum that was collected during the follicular menstrual phase of patients with endometriosis was significantly higher than that in serum from healthy control women. CONCLUSION(S): Gremlin-1 plays a role to some extent in the aberrant angiogenesis of eutopic endometrium in patients with endometriosis. It is possible that the peripheral serum level of GREM1 is a prospective serum biomarker of endometriosis.